Amplification of target nucleic acids is a fundamental method in modern molecular biology and diagnostics. Factors that adversely affect the outcome of amplification reactions include: extension of primers due to non-specific template annealing during amplification, resulting in false positives (Schlotterer and Tautz, Nucleic Acids Research, 20 (2):211-215 (1992); Ogata and Miura, Nucleic Acids Research, 26(20):4657-4661 (1998); Brukner, et al. Analytical Biochemistry, 339:345-347 (2005)); reduced amplification reaction efficiency and rate due to primer or template secondary structure; and variability of amplification due to primer dimer formation. The effects of these factors are enhanced by room temperature (RT) incubation of complete reaction mixtures prior to placement at specified reaction temperature. This would occur, for example, when large numbers of samples are prepared at one time necessitating a certain amount of sample incubation at RT. Therefore, high-throughput and diagnostic applications are often negatively impacted by reaction set-up at RT. This is a significant issue for molecular diagnostic applications, which, demands a high level of consistency and accuracy.
Various amplification methods are currently utilized in molecular diagnostics. A popular isothermal amplification diagnostic method is loop-mediated isothermal amplification (LAMP) (Notomi, et al. Nucleic Acids Research, 28(12):e63 (2000)). Typically, LAMP employs a DNA polymerase and a set of four to six synthetic primers that recognize a total of six distinct sequences on the target DNA. Recognizing six distinct sequences makes LAMP extremely specific for a target sequence. Despite the specificity of LAMP, it is adversely affected by unwanted, non-specific primer extension reactions during reaction set-up at RT.